Despite the availability of several effective antibiotics, tuberculosis (TB) remains one of the leading causes of death due to infectious disease. The treatment of Mycobacterium tuberculosis infections is difficult largely due to this organism's remarkable ability to persist in spite of months or years of antibiotic treatment, often resulting in incomplete sterilization and recurrent disease. The inevitable consequence of inadequate therapy is the emergence of multidrug-resistant strains, which are even more challenging to treat. While clinical importance of this phenomenon cannot be overstated, a mechanistic understanding of bacterial persistence during therapy and strategies to treat this infection more effectively has proven elusive. We propose to employ two new genetic methodologies to understand antibiotic persistence at the molecular level, and to identify bacterial pathways that represent targets for a more effective therapeutics. First, we will employ "transposon-site hybridization" to identify mycobacterial genes that are required for persistence during antibiotic treatment of infected animals. This information will be used to characterize the antibiotic "tolerant" state adopted by this bacterium during infection and to define novel targets for synergistic therapies. As a parallel approach, we will develop a facile system for conditionally inhibiting essential genes of M. tuberculosis in order to define pathways whose inhibition results in rapid cell death under conditions similar to the in vivo environment. We expect both of these approaches to provide insight into the metabolic state of the bacterium that is responsible for its remarkable antibiotic tolerance during infection, and to identify potential targets for drugs that could reduce the time required to treat TB. Short-course therapy could have a dramatic effect on the current TB pandemic by improving access to effective treatment, reducing the prevalence of TB, and preventing the rapid emergence of drug resistance. Tuberculosis remains a scourge largely because currently available antibiotics are effective only after many months of administration. We will pursue two independent approaches to identify bacterial pathways that could be targeted by new drugs to shorten the duration of effective treatment. Short-course therapy could impact the worldwide tuberculosis epidemic both by making treatment more widely available and by reducing the rate at which drug-resistant strains emerge. [unreadable] [unreadable] [unreadable]